槲皮素在活體及離;體抗氣喘的作用機轉
Mechanisms of Anti-Asthmatic Action of Quercetin in vivo and in vitro
中文摘要
Part I
槲皮素 (quercetin) 能有效抑制 PDE1~4，且對 PDE3 和 PDE4 具有選擇性的抑制作用，
但是否有抗氣喘的作用，是本篇實驗的目的。
我們將 BALB/c 小鼠腹腔內注射卵蛋白 (ovalbumin; OVA) ，使其敏感化，再以卵蛋白 (OVA) 氣化噴霧兩次激發 (challenge) 之。利用整體體積描述器來分析因 methacholine (MCh) 引起的氣道過度反應 (airway hyperresponsiveness response; AHR)，結果顯示 quercetin 能抑制卵蛋白 (OVA) 引起的氣道過度反應 (AHR)，quercetin (30~100
mol/kg, i.p.) 亦能劑量依存性地減少 MCh (25~50 mg/ml) 引起的 Penh 值增加，
quercetin 100 mol/kg 亦能有意義地 (P < 0.05) 抑制 MCh (12.5 mg/ml) 的 Penh 值 增加，投與 quercetin (10-100 mol/kg) 的小白鼠與敏感化未暴露於 OVA 氣化噴霧之小白 鼠 (non-treatment) 無異。觀察肺泡灌流液 (BALF) 的細胞沉澱物，發現 quercetin (10- 100 mol/kg) 也有意義地 (P < 0.05) 抑制總發炎細胞數、嗜中性血球、嗜伊紅白血球、淋巴 球及巨噬細胞。Quercetin (10-100 mol/kg) 有意義地 (P < 0.05) 降低 IL-2, IL-4, IFN-
和 TNF- 的釋放，劑量達 30-100 mol/kg 更有意義地抑制 IL-5 的釋放。Quercetin (10~100 M) 能有意義地抑制 OVA (10~100 g/ml) 累加，引起敏感化離體天竺鼠氣管的 收縮，而 quercetin (30~100 M) 亦能有意義地抑制 OVA (1 μg/ml) 引起的敏感化離體天 竺鼠氣管的收縮。由 Lineweaver-Burk 分析發現 quercetin (3~30 M) 對 PDE3 和 PDE4 呈競爭性的抑制。結論，quercetin 具選擇性且競爭性地抑制 PDE3 和 PDE4，在所用劑量 (10~100 mol/kg, i.p.) 下具抗發炎及支氣管擴張作用，可能有治療氣喘的潛力。
Part II
新型選擇性磷酸二酯酶抑制劑 (Phosphodiesterase inhibitor) 兼具消炎及支氣管擴張雙效，
有較少的副作用，所以此類藥物是氣喘治療的明日之星。所以本研究係探討 quercetin 及其衍 生物抑制 PDE 各種亞型 (isozyme) 的抑制效果，和其活性與結構的關係，希望將來合成更 有用的藥物，用以改善氣喘的治療。
本實驗室是將天竺鼠的肺臟及心臟經研磨及離心，使上清液通過Q-Sepharose 陰離子交換樹 脂，藉著改變 NaCl 的濃度，便可依序由肺臟分離得到 PDE1、PDE5、PDE2 及 PDE4，而由 心臟得到 PDE3。
而後根據 Thompson 及 Appleman 的方法，利用 cAMP 與 [3H]-cAMP 或 cGMP 與 [3H]-cGMP 作為 PDE 的受質，測定 PDE 活性。
由結果顯示 Quercetin 對 PDE3 和 PDE4 的抑制作用較強，而對 PDE1 和 PDE2 的抑制 作用較弱，但對 PDE5 無明顯的抑制作用。Ayanin 對 PDE1 及 PDE3 的抑制作用較強，但對 PDE2 及 PDE4 的抑制作用較弱。QPME 對 PDE1、PDE2、PDE3 及 PDE4 的抑制作用都很強，
而對 PDE5 的抑制作用較弱。QPA 對 PDE3 的抑制作用很強，而對 PDE1 及 PDE4 的抑制作
用較弱，但對 PDE2 及 PDE5 並無明顯抑制作用。QMPA 對 PDE3 的抑制作用非常強，而對 PDE1、PDE2 及 PDE4 的抑制作用也很強，但對 PDE5 的抑制作用較弱。綜合以上，QTME 對 PDE3 雖只有中等程度的抑制作用，但具高度的選擇性，因此有可能合成出對 PDE4 及/或 PDE3 具高度選擇性的抑制劑，果真如此，將對氣喘治療有用。
英文摘要
Part I
Quercetin effectively inhibits PDE1~4. Especially it potently inhibits PDE3 and PDE4. Whether it possesses anti-asthmatic effect is the aim of this investigation.
Female BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA), then challenged via the airway by ultrasonic nebulization of 1% OVA two times.
The airway hyperresponsiveness was measured in unrestrained animals by
barometric plethysmography using a whole-body plethysmography, and nebulized methacholin (MCh, 5~50 mg/ml). Quercetin (30~100 μmol/kg, i.p.) revealed it dose-dependently attenuated the enhanced pause (Penh) vale induced by MCh (25~50 mg/ml). Futhermore, quercetin (100 μmol/kg, i.p.) also significantly inhibited MCh (12.5 mg/ml)-induced Penh value. The Penh values of mice adminstered quercetin (10~100 μmol/kg, i.p.) did not significantly differ from those of sensitized mice without challenge by OVA (non-treatment). ? Quercetin (10~100 μmol/kg, i.p.) also significantly inhibited total inflammatory cells,
neutrophils, eosinophils, lymphocytes and macrophages, from precipitate of BALF.
It also significantly attenuated the release of IL-2, IL-4, IFN-γ and TNF-α. It at doses of 30~100 μmol/kg furthermore significantly inhibited the release of IL-5.
Quercetin (10~100 μM) significantly inhibited cumulative OVA (10~100μg/ml) induced contractions of isolated sensitized guinea pig trachea.
Quercetin (30~100 μM) further significantly inhibited OVA (1μg/ml)-induced contractions of these trchealis. Quercetin (3~30 μM) competitively inhibited PDE3 and PDE4 activities by Lineweaver-Burk analysis. In conclusion, quercetin
selectively and competitively inhibited PDE3 and PDE4 activities. At doses of 10~100 μmol/kg (i.p.), it possessed anti-inflammatory and bronchodilating effects.
Therefore it may have potential in the treatment of asthma.
Part II
New selective phosphodiesterase inhibitors have anti-inflammatory and
bronchodilating effects, but have less side effects. Therefore, these drugs will be helpful in the treatment of asthma. Aim of this study was investigation for the relationships between the structures and inhibitory effects of quercetin and its derivaties on phosphodiesterase isozymes activities. In the furture, we hope can
synthesize more useful drugs to ameliorate the treatment of asthma.
In our labolatory, we homogenized the lungs and hearts of guinea pigs, and then centrifugated. The supernatant was chromatographed over Q-sepharose, an anion exchange resin. The bound proteins (phosphodiesterases, PDEs) were eluted with various concentrations of NaCl, and separated into PDE1, PDE5, PDE2 and PDE4 in the order, whereas PDE3 was separated from hearts. Cyclic nucleotide
phosphodiesterase activitices were measured by a two-step procedure according the method of Thompson and Appleman, using cAMP with [3H]-cAMP or cGMP with [3H]-cGMP as substrate.
Quercetin potently inhibited PDE3 and PDE4, while moderately inhibited PDE1 and PDE2, but had no effect on PDE5 activity. Ayanin potently inhibited PDE1 and PDE3, whereas moderately inhibited PDE2 and PDE4, but had no effect on PDE5 avtivity. QPME potently inhibited PDE1, PDE2, PDE3 and PDE4, but moderately inhibited PDE5 activity. QPA potently inhibited PDE3, while moderately inhibited PDE1 and PDE4, but had no effect on PDE5 activity. QMPA very potently inhibited PDE3, while potently inhibited PDE1,PDE2 and PDE4, but moderatly inhibited PDE5 activity. Overall, QTME moderatly but selectively inhibited PDE3, because it had no effect on other isozymes activity. It is possible to synthesize highly selective PDE4 and/or PDE3 inhibitors, which may be useful in the treatment of asthma.
